Depigmented Centella asiatica Extraction by Pretreated with Supercritical Carbon Dioxide Fluid for Wound Healing Application
, Chiranan Khantham 2, Korawan Sringarm 1,3, Sarana Sommano 1,4 and Pensak Jantrawut 1,2Round 1
Reviewer 1 Report
Review Processes 726468
The paper compares the extraction performance from Centella asiatica leafs, using maceration with ethanol alone or combined with a “pre-treatment” of supercritical CO2 modified with ethanol. The aim was to find an extract that would not cause colour changes when added to a final product due to the deterioration of the extracted pigments. The extracts were also compared in terms of biological, antioxidant and wound healing activities. By means of a statistical analysis, the composition of the extracts in terms of triterpenoid content and its glycoside derivatives is related to these activities.
It's a work that contains a lot of data from the analysis of the extracts that are well explained. There are sufficient details given to replicate the proposed experimental procedures and analysis and there are sufficient outcome-neutral tests of the hypotheses, including positive controls and quality check. The document is well written, the figures and tables include data with statistical significance. In vitro testing puts a lot of value on the work. So the only weakness I find is the following.
What is worse explained both the methodology and the results, is the part of the tests made with CO2 and ethanol. The volume of the extractor, the amount of ethanol added, and the ratio of ethanol to CO2 are not indicated. These data should be given since the ratio of ethanol to raw material is relevant. Also, if the ethanol CO2 mixture is actually an expanded liquid phase. Theoretically, ethanol can only be said to be a modifier of CO2 if the amount with respect to CO2 is small (typically less than 10%) and solubilizes in CO2.
On the other hand, this treatment is actually another extraction stage prior to ethanol maceration. It would be very interesting if, based on the density, polarity, pH and other properties of this solvent mixture, the elimination of the pigments in the leaf pieces could be explained.
Adding supercritical CO2 is a step that forces work under pressure and therefore will increase operating and installation costs. Therefore, it must be well justified in practical and theoretical terms. It must give a relevant advantage that cannot be obtained by using a sequence of two macerations with ethanol, one of them one of them with reduced pH (as will probably cause the CO2). Note that pigments are low soluble in supercritical CO2.
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Reviewer 2 Report
Page 3, 2.3.1 Conventional extraction - The quantities of grams / kilograms of powder of C. asiatica used for the extraction should be given.
Page 3, 2.3.2. Pre-treatment using scCO2 - Why was 85% instead of 70% ethanol used for maceration?
Were the extracts (CV, SCE1, SCE2) evaporated to dryness or only concentrated?
Page 4 line 3 – “inter-spot” change to „inter-band”
Page 4 line 12 – should be “the following settings: detector mode, automatic; slit dimensions, …..” The dimensions of the slit are given in wrong units, they should be changed to millimeters. For bandwise applied samples the slit length should be 70-80% of the band length. Why was the smaller slit length used?
Please complete the following information: measurement mode and wavelength for plates scanning. How many microliters of samples and standards were applied on plates?
Page 4, 2.5.2 HPLC - The symbol descriptions for Equation 1 are unclear. In the explanations of the symbols under the equation, it was written: "Rs = peak areas of asiaticoside in the standard solution A, Cs = concentration of USP RS asiaticoside in the standard solution A (mg / mL)". On page 3 in chapter 2.4.1, the authors wrote: "The asiaticoside United States Pharmacopoeia reference standard (USP RS) was dissolved in methanol at 0.5 mg / mL for standard solution A." Was an 0.05 mg / mL or 0.5 mg / mL asiaticoside solution used for HPLC analysis ? Moreover, the wording "sample stock solution" should be explained. Please specify which solution is this?
Page 5, line 3 - Please, enter the concentration of ethanolic DPPH solution.
Page 7, chapter 3.2., line 3 - word “lane” change to “track”
Page 9 chapter 4.4 , line 4-5 – “The Trolox standard curve in the ABTS+ scavenging assay was y = - 7.547 x = 1.4813” What does "= 1.4813" mean in this calibration curve equation?
Page 12 Table 3 - Please check and correct all the results in Table 3 because they are completely absurd and inconsistent with section 3.5.2. Cell migration and angiogenesis activity e.g. 6.±50.4322, 1.± 59.8385, 6.03±45.75.
Author Response
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Reviewer 3 Report
The work is interesting and complete.
I think that the authors must do a new bibliographic revision. There are new researches on supercritical carbon dioxide extraction, DPPH and bioactive compounds, etc., in the last year. These work should be include in a work that it is going to be published. In addition, it could improve the discussion.
Author Response
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